Secretory protein

ABSTRACT

The disclosure of the present application relates to a secretory deleted split hand/split foot 1 (sDSS1) protein, the amino acid sequence thereof, the nucleic acid sequence thereof, and the applications of the same. The sDSS1 protein is a secretory protein from higher primate, and can be detected in human serum and cerebral spinal fluid (CSF). The sDSS1 protein can form conjugate with oxidized protein under nonenzymatic condition or with amyloid-beta (Aβ) polypeptide to reduce formation of Aβ oligomer. The addition of sDSS1 protein to culture medium can shield the cytotoxicity induced by oxidized protein, Aβ oligomer, amylin oligomer and glycosylated protein, so as to protect the cells against these toxoproteins. The sDSS1 protein can prolong survival time of senescence-accelerated mice significantly. The protein can be used to prevent and treat the diseases induced by oxidized protein, glycated protein, Aβ protein accumulation, amylin protein accumulation or excessive formation or accumulation of other pathogenic proteins with similar features, and has important potential in biological medicine.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. Non-Provisional patentapplication Ser. No. 17/028,769, filed Sep. 22, 2020, which is adivision of U.S. Non-Provisional patent application Ser. No. 16/145,251,filed Sep. 28, 2018, now U.S. Pat. No. 10,815,282, which iscontinuation-in-part application of PCT Application No.PCT/CN2017/090785 filed on Jun. 29, 2017, which claims the benefit ofChinese Patent Application No. 201610519038.9 filed on Jul. 4, 2016, thedisclosure of which is hereby incorporated by reference in theirentirety.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy created on Nov. 5, 2021, isnamed 57275701301 SL.txt and is 14,615 bytes in size.

BACKGROUND

The present application relates generally to a secretory protein, thesecretory protein can be used to prepare the drugs for preventing andtreating the diseases induced by excessive formation or excessiveaccumulation of junk proteins.

DESCRIPTION OF RELATED ART

In normal physiological activities, the organism generates lots of junkproteins, including oxidized protein, glycosylated protein and someabnormal spliced proteins (polypeptide). The organism retains multiplemechanisms for removing junk proteins to maintain normal physiologicalfunction. However, the aging or diseases will induce excessive formationof junk proteins or degrade the organism's ability to remove junkproteins, so that lots of junk proteins accumulate. The abnormalaccumulation of junk proteins inside or outside the cells is the keymechanism inducing a series of diseases. The typical diseases includechronic kidney disease, Alzheimer's disease (AD), Huntington's disease,diabetes complications and so on^([1-5]). The accumulation of oxidizedprotein, glycated protein or other junk proteins in the circulatorysystem is one of the key causes for the aging of organism^([6-7]). It isproved by research that the advanced oxidation protein products (AOPP)in the serum damages renal cells, and it is the main pathogenesis ofchronic kidney disease. The AOPP in serum can induce the programmedapoptosis of islet β cells^([3-8]). The β amyloid hypothesis indicatesthat the synaptic dysfunction and neuron death resulted from progressiveaccumulation of toxoprotein induced by unbalance of generation andremoval of Aβ protein in tissues are the first causes of AD ^([9]). Theamylin protein not only performs abnormal aggregation in the insulartissues of partial diabetics, but also exists in the plaques of braintissue, and it is closely related to the progress of diabetes andneurodegenerative diseases^([10, 11]). Based on these findings, in somedisease models, the Aβ aggregation or formation in the AD animal patternis blocked by using antibody^([12]), polypeptide drug^([13]) ormicromolecular drug^([14]), the formation of neuronal tissue plaques canbe reduced, and the animal cognition level is increased. These resultsshow that using drugs to depress the formation and aggregation of thesepathogenic proteins or to promote the removal of pathogenic proteins toreduce the accumulation of pathogenic proteins is an important method toprevent or treat these diseases.

Previous research indicates that when the oxidative stress occurs in thecell, the DSS1 (deleted split hand/split foot 1) protein, as a highlyconservative small protein in eukaryote, can be covalently modified tooxidized protein under the conditions of enzymatic reaction and ATPconsumption, such a modification will mediate the oxidized protein todegrade in the cell^([15]). The DSS1 gene knockout leads to cell death;the cells with high expression of DSS1 protein manifest significantresistance to the oxidative stress or antineoplastic-induced cellapoptosis^([16]). These results show the vital function of DSS1 proteinin the course of removing oxidized protein from cells, and it is the keyto the existence of cells.

The related references are described below:

-   1. Dobson C M (1999) Protein misfolding, evolution and disease.    Trends Biochem Sci 24:329-332.-   2. Liang M, Wang J, Xie C, Yang Y, Tian J W, Xue Y M, Hou F F (2014)    Increased plasma advanced oxidation protein products is an early    marker of endothelial dysfunction in type 2 diabetes patients    without albuminuria 2. J Diabetes 6(5):417-26.-   3. Cao W, Hou F F, Nie J (2014) AOPPs and the progression of kidney    disease. Kidney Int Suppl (2011) 4(1):102-106.-   4. Sadigh-Eteghad S, Sabermarouf B, Maj di A, Talebi M, Farhoudi M,    Mahmoudi J (2015) Amyloid-beta: a crucial factor in Alzheimer's    disease. Med Princ Pract 24(1):1-10.-   5. Choe Y J, Park S H, Hassemer T, Korner R, Vincenz-Donnelly L,    Hayer-Hartl M, Hartl F U (2016) Failure of RQC machinery causes    protein aggregation and proteotoxic stress. Nature 531(7593):191-5.-   6. Ott C, Grune T (2014) Protein oxidation and proteolytic    signalling in aging. Curr Pharm Des 20(18):3040-51.-   7. Simm A, Müller B, Nass N, Hofmann B, Bushnaq H, Silber R E,    Bartling B (2015) Protein glycation—Between tissue aging and    protection. Exp Gerontol 68:71-5.-   8. Liang M, Li A, Lou A, Zhang X, Chen Y, Yang L, Li Y, Yang S, Hou    F F (2017) Advanced oxidation protein products promote NADPH    oxidase-dependent β-cell destruction and dysfunction through the    Bcl-2/Bax apoptotic pathway. Lab Invest 24. [Epub ahead of print].-   9. Zhao L N, Long H, Mu Y, Chew L Y (2012) The toxicity of amyloid β    oligomers. Int J Mol Sci 13(6):7303-27.-   10. Fernandez M S (2014) Human IAPP amyloidogenic properties and    pancreatic β-cell death. Cell Calcium 56(5):416-27.-   11. Lim Y A, Rhein V, Baysang G, Meier F, Poljak A, Raftery M T,    Guilhaus M, Ittner L M, Eckert A, Götz J (2010) Abeta and human    amylin share a common toxicity pathway via mitochondrial    dysfunction. Proteomics 10 (8): 1621-33.-   12. Winblad B, Andreasen N, Minthon L, Floesser A, Imbert G,    Dumortier T, Maguire R P, Blennow K, Lundmark J, Staufenbiel M,    Orgogozo J M, Graf A (2012) Safety, tolerability, and antibody    response of active Aβ immunotherapy with CAD106 in patients with    Alzheimer's disease: randomised, double-blind, placebo-controlled,    first-in-human study. Lancet Neurol 11(7):597-604.-   13. Chang L, Cui W, Yang Y, Xu S, Zhou W, Fu H, Hu S, Mak S, Hu J,    Wang Q, Ma V P, Choi T C, Ma E D, Tao L, Pang Y, Rowan M J, Anwyl R,    Han Y, Wang Q (2015) Protection against β-amyloid-induced synaptic    and memory impairments via altering β-amyloid assembly by    bis(heptyl)-cognitin. Sci Rep 5:10256.-   14. Kim H Y, Kim H V, Jo S, Lee C J, Choi S Y, Kim D J, Kim Y (2015)    EPPS rescues hippocampus-dependent cognitive deficits in APP/PS1    mice by disaggregation of amyloid-β oligomers and plaques. Nat    Commun 6:8997.-   15. Zhang Y, Chang F M, Huang J, Junco J J, Maffi S K, Pridgen H I,    Catano G, Dang H, Ding X, Yang F, Kim D J, Slaga T J, He R, Wei S    J (2014) DSSylation, a novel protein modification targets proteins    induced by oxidative stress, and facilitates their degradation in    cells. Protein Cell 5(2):124-40.-   16. Rezano A, Kuwahara K, Yamamoto-Ibusuki M, Kitabatake M,    Moolthiya P, Phimsen S, Suda T, Tone S, Yamamoto Y, Iwase H,    Sakaguchi N (2013) Breast cancers with high DSS1 expression that    potentially maintains BRCA2 stability have poor prognosis in the    relapse-free survival. BMC Cancer 13:562.

SUMMARY OF THE APPLICATION

In the latest study, we (inventors) have found that there is a newsubtype of DSS1 protein in higher primate (anthropoid subfamily) genome,named secretory DSS1 protein (sDSS1). The sDSS1 is the first DSS1protein subtype discovered, and its sequence, properties and functionare highly similar to DSS1. However, it can be secreted into blood andcerebral spinal fluid, its properties are more active, and can form aconjugate with the oxidized protein in serum or buffer solution withoutenergy-consuming enzymatic reaction or combine with Aβ protein andreduce the formation of Aβ oligomer. The sDSS1 protein added to theculture medium can shield the cytotoxicity induced by oxidized protein,Aβ oligomer, amylin oligomer and glycosylated protein to protect cellviability. Therefore, we identify this new type of protein sDSS1 as apromising drug for preventing and treating the diseases induced byoxidized protein, glycosylated protein, Aβ, amylin and other pathogenicproteins with similar features.

The specific technical solution is described below:

A sDSS1 protein is provided, which may have an amino acid sequence ofhuman protein sDSS1 as shown in SEQ ID NO: 1. A protein having the sameor similar amino acid sequence as SEQ ID NO: 1 exists in theAnthropoidea animals.

Preferably, the Anthropoidea animals may further be chimpanzee, bonobo,gorilla, orangutan, white-cheeked gibbon, golden snub-nosed monkey,rhesus macaque, olive baboon, Angola colobus, sooty mangabey, drill andnorthern pigtail macaque; wherein Pan troglodytes (a chimpanzee) sDSS1protein has an amino acid as set forth in SEQ ID NO: 5, Pan paniscus (abonobo) sDSS1 protein has an amino acid as set forth in SEQ ID NO: 6,Gorilla gorilla (a gorilla) sDSS1 protein has an amino acid as set forthin SEQ ID NO: 7, Pongo abelii (an orangutan) sDSS1 protein has an aminoacid as set forth in SEQ ID NO: 8, Nomascus leucogenys (a white-cheekedgibbon) sDSS1 protein has an amino acid as set forth in SEQ ID NO: 9,Rhinopithecus roxellana (a golden snub-nosed monkey) sDSS1 protein hasan amino acid as set forth in SEQ ID NO: 10, Macaca mulatta (a rhesusmacaque) sDSS1 protein has an amino acid as set forth in SEQ ID NO: 11,Papio anubis (an olive baboon) sDSS1 protein has an amino acid as setforth in SEQ ID NO: 12, Colobus angolensis (a Angola colobus sDSS1protein has an amino acid as set forth in SEQ ID NO: 13, Cercocebus atys(a sooty mangabey) sDSS1 protein has an amino acid as set forth in SEQID NO: 14, Mandrillus leucophaeus (a drill) sDSS1 protein has an aminoacid as set forth in SEQ ID NO: 15, Macaca nemestrina (a northernpigtail macaque) sDSS1 protein has an amino acid as set forth in SEQ IDNO: 16.

Preferably, the sDSS1 protein includes a N-terminal amino acid sequenceof 58 amino acids and a C-terminal amino acid sequence of 31 aminoacids, wherein the human sDSS1 protein has a N-terminal amino acidsequence of 58 amino acids as set forth in SEQ ID NO: 3, the human sDSS1protein has a C-terminal amino acid sequence of 31 amino acids as setforth in SEQ ID NO: 2; wherein the N-terminal amino acid sequence of the58 amino acids includes 3 or more amino acid sequences with consecutiveacidic amino acids, each of amino acid sequences with consecutive acidicamino acids includes no more than 10 acidic amino acids, any twoadjacent amino acid sequences of the amino acid sequences withconsecutive acidic amino acids have a spacing of no more than 4 aminoacids, and the spacing includes at least one hydrophobic amino acid, apH value is not higher than 4.5, the N-terminal amino acid sequence ofthe 58 amino acids includes no less than 10 acidic amino acids; theC-terminal amino acid sequence following position 58 of the N-terminalamino acid sequence of the 58 amino acids are relatively hydrophobicoverall, the C-terminal amino acid sequence of the 31 amino acidsincludes no less than 10 hydrophobic amino acids;

wherein the hydrophobic amino acid is selected from the group consistingof alanine, isoleucine, leucine, valine, cysteine, phenylalanine,methionine, tryptophan, and tyrosine,

the neutral amino acid is selected from the group consisting ofthreonine, glycine, serine, histidine, and glutamine;

the acidic amino acid is selected from the group consisting of glutamicacid, aspartate, proline, and asparaginate; and

the basic amino acids is selected from the group consisting of arginine,and lysine.

Preferably, the sDSS1 protein in the Anthropoidea animals includes aC-terminal amino acid sequence of:

-   -   X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉X₃₀X₃₁;

X₁ is a neutral amino acid; X₂ is a hydrophobic amino acid; X₃ and X₄are hydrophobic amino acids; X₅ is a hydrophobic amino acid; X₆ is ahydrophobic amino acid; X₇ is a hydrophobic amino acid; X₈ is ahydrophobic amino acid; X₉ is a hydrophobic amino acid; X₁₀ is an acidicamino acid; X₁₁ is a neutral amino acid; X₁₂ is a hydrophobic aminoacid; X₁₃ is a hydrophobic amino acid; X₁₄ is a neutral amino acid; X₁₅is a hydrophobic amino acid; X₁₆ is a hydrophobic amino acid; X₁₇ is ahydrophobic amino acid; X₁₈ is a hydrophobic amino acid; X₁₉ is a basicamino acid; X₂₀ is an acidic amino acid; X₂₁ is a basic amino acid; X₂₂is a neutral amino acid; X₂₃ is a basic amino acid; X₂₄ is a hydrophobicamino acid; X₂₅ is a hydrophobic amino acid; X₂₆ is a neutral aminoacid; X₂₇ is a hydrophobic amino acid; X₂₈ is a hydrophobic amino acid;X₂₉ is a hydrophobic amino acid; X₃₀ is a hydrophobic amino acid; andX₃₁ is a hydrophobic amino acid;

an amino acid sequence having 40% or more homology to the C-terminalamino acid sequence of the 31 amino acids, wherein the amino acidsequence has a same or similar property and function to a C-terminalamino acid sequence of a human sDSS1 protein.

A polypeptide comprising an amino acid sequence constructed based on theN-terminal 58 amino acid sequence and the C-terminal 31 amino acidsequence of the sDSS1 protein as described hereinabove, wherein

1) the polypeptide sequence has a N-terminal having 40% or more homologyto the N-terminal amino acid sequence of the 58 amino acids, and thepolypeptide sequence has a C-terminal having 40% or more homology to theC-terminal amino acid sequence of the 31 amino acids, a protein encodedby the polypeptide sequence has a same or similar property and functionto a human sDSS1 protein; or

2) a N-terminal of the polypeptide sequence is based on a N-terminalamino acid sequence of 58 amino acids of a human sDSS1 protein, or is asequence having 40% or more homology to the N-terminal amino acidsequence of the 58 amino acids of the human sDSS1 protein, wherein aC-terminal or the N-terminal of the polypeptide is fused with otheramino acid sequence, the other amino acid sequence for fusion has anidentical or similar property to a C-terminal amino acid sequence of 31amino acids of the human sDSS1 protein and perform the same or similarfunctions, a modified protein encoded by the polypeptide sequenceperforms an identical or similar function to the human sDSS1 protein; or

3) the peptide sequence is constructed by fusing the C-terminal aminoacid sequence of the 31 amino acids in the sDSS1 protein, such as isdescribed hereinabove, with other polypeptide sequence.

The fusion protein includes a full sequence or a partial sequence of thesDSS1 protein such as is described hereinabove, and the polypeptidesequence such as is described hereinabove.

Preferably, the fusion protein is a protein complex formed by linkingthe protein sDSS1 protein, a carrier protein, an antibody or otherarbitrary amino acid sequence.

A complex includes a full sequence or a partial sequence of the sDSS1protein such as is described hereinabove, the polypeptide sequence suchas is described hereinabove, or a full sequence or a partial sequence ofthe fusion protein such as is described hereinabove.

Preferably, the complex is a complex formed by linking the sDSS1 proteinto a pharmaceutically acceptable drug carrier.

Preferably, the pharmaceutically acceptable drug carrier includes one ormore of a microsphere/capsule, liposome, micro-emulsion, nanoparticle,magnetic particle and gel.

A nucleotide encodes the sDSS1 protein such as is described hereinabove,or the polypeptide such as is described hereinabove.

Preferably, the nucleotide includes DNA and RNA.

A cell expresses the sDSS1 protein such as is described hereinabove orthe polypeptide such as is described hereinabove.

Preferably, the cell is a stem cell, a precursor cell or an adult cellof a mammal.

Preferably, the mammal is a human, an orangutan, a monkey, a horse, acattle, a sheep, a pig, a donkey, a dog, a rabbit, a cat, a rat or amouse.

Preferably, the cell includes an embryo stem cell, an inducedmultipotential stem cell or a stem cell derived from a primary culture,a multipotential or monopotential stem cell derived from a mother celldifferentiation.

An expression system, wherein a nucleotide sequence encoding the sDSS1protein such as is described hereinabove or the polypeptide such as isdescribed hereinabove is introduced into an organism, and the sDSS1protein such as is described hereinabove or the polypeptide such as isdescribed hereinabove is expressed in the organism.

Preferably, the expression system is selected from the group consistingof eukaryotic expression plasmid vector, adenovirus, slow virus,retrovirus, CRISPR/Cas technique and other feasible gene-editingtechniques.

Preferably, the organism is a human, an orangutan, a monkey, a horse, acattle, a sheep, a pig, a donkey, a dog, a rabbit, a cat, a rat, amouse, a chicken, a duck or a goose.

A drug primarily targets the sDSS1 protein such as is describedhereinabove or the polypeptide such as is described hereinabove, whereinthe drug can affect an expression level of the sDSS1 protein such as isdescribed hereinabove or the polypeptide such as is describedhereinabove in the organism upon administration.

Preferably, the drug is a chemical micromolecular drug, aprotein/polypeptide drug, a nucleic acid drug, or a nanodrug.

Preferably, the nucleic acid drug includes one or more of a siRNA, amicroRNA, an antisense oligonucleotide, a triple strand DNA and aribozyme.

A method of producing a protein, includes the following steps:

S1. constructing an expression vector: inserting a nucleotide sequencecoding the sDSS1 protein such as is described hereinabove or thepolypeptide such as is described hereinabove into a plasmid andintroducing the plasmid into bacteria or yeast cell, or inserting thenucleotide sequence coding the sDSS1 protein such as is describedhereinabove or the polypeptide such as is described hereinabove intogenome of an insect cell or a mammalian cell;

S2. expressing the sDSS1 protein: expanding a culture of the bacteria,yeast cell, insect cell or mammalian cell as modified in S1, andcollecting a culture medium or cell lysate containing the sDSS1 proteinsuch as is described hereinabove or the polypeptide such as is describedhereinabove;

S3. purifying the sDSS1 protein: coarse filtering and purifying theculture medium or cell lysate obtained in S2 to obtain the sDSS1protein.

A method of producing a protein, includes using chemical synthesistechnique to produce the sDSS1 protein such as is described hereinaboveor the polypeptide such as is described hereinabove.

A method of producing a protein, includes using in vitro ribosomeexpression system to produce the sDSS1 protein such as is describedhereinabove or the polypeptide such as is described hereinabove.

A method of diagnosing, preventing or treating disease, includespreparing a diagnostic reagent, a preventive drug, or a therapeutic drugusing the sDSS1 protein, polypeptide, fusion protein, complex,nucleotide sequence, cell, expression system, or drug such as isdescribed hereinabove, and administering the diagnostic reagent,preventive drug, or therapeutic drug to a subject in need thereof.

Preferably, the disease is a disease induced by excessive formation oraccumulation of pathogenic protein/polypeptide.

Preferably, the pathogenic protein/polypeptide is an oxidized proteinproduct, glycosylated protein product, an amyloid precursor protein anda spliceosome thereof, an islet amyloid polypeptide and a spliceosomethereof, or other pathogenic protein/polypeptides having featuressimilar to an oxidized protein a glycosylated protein, an amyloidprotein or an islet amyloid polypeptide.

Preferably, the diagnosing of the disease includes detecting one or moreof an expression level of a full or partial sequence of the amino acidsequence, mRNA level and number of gene copies of the sDSS1 protein suchas is described hereinabove.

Preferably, the preventing includes one or more of genetic modification,nucleic acid introduction, drug injection/administration, cellulartransplantation and tissue transplantation.

Preferably, the treating includes one or more of genetic modification,nucleic acid introduction, drug injection/administration, cellulartransplantation and tissue transplantation.

The characteristics and/or beneficial effects of the present applicationare:

1. The polypeptide sequence of the sDSS1 protein and typical human sDSS1protein provided by the present application isMSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNWDDDNVEDDFSNQLRATVLLMILVCETPYGCYVLHQKGRMCSAFLCC (see SEQ ID NO: 1). According tobioinformatic analysis, the protein is a protein of anthropoid subfamilyanimals.

2. According to bioinformatic analysis and cell experiment, the 31-aminoacid carbon terminal sequence of the sDSS1 protein is a signal peptideand has critical effect on the properties and secretion property of theprotein. The C-terminal sequence of the 31 amino acids isTVLLMILVCETPYGCYVLHQKGRMCSAFLCC (see SEQ ID NO: 2).

3. The sDSS1 protein defined in the present application can be combinedwith oxidized protein, glycosylated protein, Aβ protein and amylinprotein and shield the cytotoxicity induced by aggregation of thesetoxoproteins, so it has important potential in treating the diseasesinduced by excessive formation or excessive accumulation of thesetoxoproteins and other pathogenic proteins with similar features.

4. The sDSS1 protein of the present application is produced byfermentation of Escherichia coli. The nucleotide sequence coding thesDSS1 protein is inserted into pET151D plasmid, during sDSS1 expression,the N-terminal is fused with a 6-his tag and a V5 tag for purificationand immunoblotting detection. The expression of protein in Escherichiacoli is preliminarily purified by using Ni-NTA gel column, and then theSDS-PAGE is used for gel purification. The cut strip of gel containingHis-V5-sDSS1 protein is put in a dialysis bag containing transferbuffer, the protein is extracted from the gel under the drive ofelectric field and collected in the dialysis bag. The protein purifiedby the SDS polyacrylamide gel electrophoresis analysis can reach thelevel for bioexperiment.

5. According to molecular experiment, the sDSS1 protein of the presentapplication can be combined with the oxidized protein in serum and theoxidized protein in buffer solution to form conjugates, or combine withAβ protein to reduce the formation of Aβ oligomer.

6. The cell experiment proves that the sDSS1 protein of the presentapplication can shield the cytotoxicity induced by oxidized protein,glycosylated protein, Aβ oligomer and amylin oligomer in the culturemedium effectively, so as to maintain the cell viability.

To sum up, the present application provides a sDSS1 protein, thebiological property and activity of sDSS1 protein are proved by theresearch in bioinformatics, molecular biology and cellular biology. ThesDSS1 protein can reduce the cytotoxicity induced by oxidized protein,glycosylated protein, Aβ oligomer and amylin oligomer in culture mediumeffectively to maintain cell viability. As the sDSS1 protein is acongenital protein of higher primate, it is free of immunoreaction inclinical application. Therefore, the present application provides acandidate drug for preventing and treating the diseases induced byexcessive formation or excessive accumulation of oxidized protein,glycosylated protein, Aβ protein, amylin polypeptide and otherpathogenic proteins with similar features, and it has importantapplication prospects in biomedicine.

BRIEF DESCRIPTION OF THE DRAWINGS

The present application is further explained by the following attachedfigures, so as to make the present application clear and complete, butnot to limit the scope of protection of the present application.

FIG. 1A. illustrates the comparison between human DSS1 gene cDNA andhuman sDSS1 gene cDNA.

The sDSS1 gene is a new subtype of DSS1 gene, the comparison betweenhuman DSS1 gene cDNA (NM 006304.1, 509 bp) and human sDSS1 gene cDNA(AK309241.1, 1195 bp) shows an overlapping area, the nucleic acidsequence of the overlapping area can encode N-terminal 58 amino acidsequences according to analysis.

FIG. 1B. illustrates the comparison of The sDSS1 protein amino acidsequences of 13 species of primates.

The sDSS1 protein amino acid sequences of 13 species of primates werecompared by using Clustal X2.1 software, the results show that the sDSS1protein amino acid sequence is highly conservative, N-terminal 58 aminoacid sequences are identical, and the C-terminal 31 amino acid sequenceshave point mutation only at a few sites.

FIG. 2A. illustrates the GFP protein distribution.

In plasmid transfected 293T cell, the GFP protein distribution wasobserved 24 h later. The green fluorescence in control cell (GFP) wasclear and bright, and the background in solution was dim. Obvious greenfluorescence signal was observed in the culture solution of sDSS1 andGFP chelated protein (sDSS1-GFP) or sDSS1 protein C-terminal 31 aminoacid sequences and GFP chelated protein (sDSS1-c-GFP), and theintracellular fluorescence disperses and the intensity declines, meaningthat the GFP protein was taken out of the cell with the sDSS1 protein orsDSS1 protein C-terminal sequence secretion.

FIG. 2B. illustrates that the sDSS1 protein is a secretory protein.

Point membrane immunoblotting tests for detecting transfected cellculture medium, the results show that the GFP signal was detected insDSS1-GFP and sDSS1-c-GFP culture media, and there was no obvious signaldetected in blank control and GFP control group, proving that the sDSS1protein is a secretory protein, and C-terminal 31 amino acid sequencesare signal peptide.

FIG. 3A. illustrates that the sDSS1 signal can be detected in the humanserum or human cerebral spinal fluid (CSF) sample.

The sDSS1 signal can be detected in the human serum or human cerebralspinal fluid (CSF) sample by using specific antibody of sDSS1 proteinC-terminal polypeptide sequence (antigen sequence: C-terminal 31 aminoacid sequences of sDSS1 protein). The Human CSF sample was from seniorcitizens, the serum sample a was from the blood of a youth afterstrenuous exercise, the serum samples b, c and d were from the blood ofyouths in resting state.

FIG. 3B. illustrates the specific mRNA sequence of sDSS1 gene.

The specific mRNA sequence of sDSS1 gene (amplified product is 293 bp)can be detected in human astrocytomas glioblastoma (U-87 MG) by usingPCR, the DSS1 gene is used as control (amplified product is 238 bp).

FIG. 4A-FIG. 4B. illustrates that coomassie brilliant blue stainingshows the content of objective protein in the sDSS1 protein productionand purification processes.

FIG. 4A. The positive Escherichia coli cloning strain was selected toexpand culture, the addition of IPTG can induce the expression of sDSS1protein, the expression level of objective protein in the cell withoutinduction was very low. FIG. 4B. The concentrated lysate afterpreliminary purification of Ni-NTA gel column and the objective proteincontent after purification were tested, channel a shows the purifiedsDSS1 protein, channel b shows the preliminarily purified cell lysissolution.

FIGS. 5A-5D. illustrates that biochemical experiment and cell experimentprove that the sDSS1 protein can combine with oxidized protein andshield the toxicity of oxidized protein.

FIG. 5A. The 0.72 μg purified sDSS1 protein was mixed with differentproportions of serum protein for incubation, the sDSS1 protein wastested by V5 conjugated protein (V5-HRP), the result shows that thesDSS1 protein and oxidized protein of serum formed macromolecularprotein complex.

FIG. 5B. The AOPP (200 μg/mL) and the purified sDSS1 proteins atdifferent concentrations were incubated at 4° C. over night, the productwas separated by SDS-PAGE, and the Coomassie brilliant blue stainingshows that the sDSS1 protein and AOPP can form macromolecular complex,the complex content increases with sDSS1 protein concentration. FIG. 5C.The culture medium is mixed with 10% oxidized serum, the cellproliferation was reduced significantly, the sDSS1 protein in culturemedium can shield the cytotoxicity derived from the oxidized serum. FIG.5D. The culture medium without serum was mixed with 100 μg/mL AOPPprotein to reduce the cell viability, the addition of sDSS1 protein atisoconcentration can retrieve cell viability, the 100 μg/mL BSA was usedfor control group. The data was analyzed by t-test two-tailed test andvalidated by ANOVE. **, p-value<0.01.

FIGS. 6A-6E. illustrates that the sDSS1 protein reduces the formation ofAβ oligomer, and reduces the cytotoxicity and cell apoptosis induced byAβ oligomer.

FIG. 6A. Different proportions of sDSS1 protein were mixed with 10 μg Aβprotein before incubation, according to Aβ antibody test, the sDSS1protein and Aβ formed covalently conjugated high molecular weightprotein complex, such a conjugation can reduce the formation of Aβoligomer with cytotoxicity. FIG. 6B. V5 sDSS1 protein was tested byconjugated protein (V5-HRP), the result shows that the sDSS1 protein andAβ formed a protein complex. FIG. 6C. The addition of Aβ oligomer to theculture medium induced cytotoxicity, the cell viability was degraded,the sDSS1 protein can shield the cytotoxicity induced by Aβ oligomercompletely. FIG. 6D. The cell apoptosis experiment shows that the sDSS1protein added to the culture medium reduced the early apoptosis and lateapoptosis of SH-SY5Y cells induced by Aβ oligomer significantly, so asto reduce the effect of toxoprotein on cells. FIG. 6E. The sDSS1 proteincan shield the toxicity of Aβ oligomer for mouse nerve stem cells(NSCs). The data was analyzed by t-test two-tailed test and validated byANOVE. **, p-value<0.01.

FIG. 7. illustrates that the addition of sDSS1 protein can retrieve thecell viability, promoting the cell survival.

The amylin oligomer added to the culture medium induces cytotoxicity andreduces cell viability, the addition of sDSS1 protein can retrieve thecell viability, promoting the cell survival. The data was analyzed byt-test two-tailed test and validated by ANOVE. **, p-value<0.01.

FIG. 8. illustrates that the cell viability decline can be retrieved bysDSS1 protein.

The cell viability decline induced by 400 μg/mL glycosylated protein canbe retrieved by sDSS1 protein, the retrieving effect increased with thesDSS1 protein concentration (100 μg/mL to 200 μg/mL). The 400 μg/mL BSAprotein was used for control group. The data was analyzed by t-testtwo-tailed test and validated by ANOVE. **, p-value<0.01.

FIG. 9A. illustrates that Operating method of injecting virus into thelateral ventricle of SAMP8 mouse and virus injection site.

FIG. 9B. illustrates that the survival rate after operation of the mouseinjected with adenovirus expressing sDSS1 protein was apparently higherthan that of control mouse.

The adenovirus was injected into the lateral ventricle of a 5 months oldsenescence-accelerated mouse SAMP8 mouse (1 μL, virus into right andleft brains respectively), the animal survival was observedcontinuously. The result shows that the survival rate after operation ofthe mouse injected with adenovirus expressing sDSS1 protein wasapparently higher than that of control mouse (expressing GFP protein).The data was analyzed by ANOVE. **, p-value<0.01.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The preferred solutions of the present application are described andvalidated with examples in the following text, not to limit the scope ofthe present application. All scope of the present application aresubject to the scope of the Claims.

The experimental methods for the following cases are conventionalexperimental methods unless otherwise specified.

In the following embodiments, the sDSS1 protein was produced in-houseand its purity reached the level for bioexperiment, the other materialsand reagents were commercially available.

Example 1, sDSS1 Protein is a Secretory Protein from Primate

Bioinformatic Analysis Tool:

National Center of Biotechnology Information (NCBI) genome database;Nucleotide blast tool (NCBI); Align sequences nucleotide blast tool(NCBI), Translate tool (SIB Bioinformatics Resource Portal); ClustalX2.1: Multiple Sequence Alignment (EMBL-EBI); SecretomeP 2.0 (CBSprediction service); WoLF PSORT II.

Bioexperimental Method:

1. Cell culture, the 293T cells were bought from American type culturecollection (ATCC), the cells were cultured in the cell culture mediumcontaining 90% basal medium (Dulbecco's modified eagle medium, DMEM)(Life technology C#12500062) and 10% Fetal bovine serum (FBS) (GibcoC#10100-147), cultured in cell incubator (temperature 37° C., humidity95%, CO2 concentration 5%), subcultured once every two days.

2. Cell transfection, the 293T cells were inoculated in a 6-well plateas per 3×10⁵ per well, mixed with 1.5 mL cell culture medium, theplasmid was transfected when the cells have been adhering to the wallfor 12 hours. The eukaryotic expression plasmid pCMV-C-Flag was used,the inserted nucleic acid sequence expressing sDSS1 protein wasexpressed as SEQ ID NO: 17. 2500 ng of the plasmid was diluted and mixedwith 750 uL Opti-MEM®Medium (Life technology C#31985062) uniformly, 10μL transfection reagent Lip2000 (Invitrogen C#12566014) was diluted andmixed with 7504, Opti-MEM®Medium uniformly, the diluted plasmid solutionwas instilled into the diluted transfection reagent drop by drop, mixeduniformly and incubated at normal temperature for 5 minutes. The cellculture medium was blotted from the 6-well plate, the cells were cleanedwith PBS, and then the incubated transfection working fluid was applied.The cells were cultured in the incubator continuously, the fluorescentprotein expression of the cells 2 was observed during 24 to 48 hours.

3. Western blotting, the PVDF membrane was activated by methanol anddried, the control culture medium and different transfection cellculture media were dripped onto the membrane. When the membrane wasdried, the PVDF membrane completed 1% BSA sealing, primary antibody(Rabbit-anti-GFP) (Cell signal technology C#2956) incubation, secondaryantibody (Goat-anti-rabbit HRP antibody) (Zsbio, ZDR-5403) incubation inturn. The membrane was cleaned with PBST three times, developed byluminescent liquid (Zsbio, ZLI-9017) and the bands were exposed by X-rayfilm.

Result Analysis:

In the bioinformatic analysis of shfml gene in human genome, it wasfound that the gene has multiple transcripts (see shfml gene informationin NCBI database, http://www.ncbi.nlm.nih.gov/gene/7979). Besides anmRNA sequence of jointly coded DSS1 protein sequence (NM 006304.1, 509bp), there is a longer mRNA sequence (AK309241.1, 1195 bp). The shortmRNA sequence and long mRNA sequence only have 256 bp repeat sequence(FIG. 1A). According to nucleic acid sequence analysis, it can be seenfrom the Translate tool that the repeat sequence can encode DSS1 proteinN-terminal 58 amino acid sequences. The long mRNA sequence coded for 89amino acids. According to the alignment of polypeptide sequences, thelong mRNA encoded polypeptide sequence and DSS1 polypeptide sequencehave the overlapping area of N-terminal 58 amino acids, and thevariation area of 31 amino acids. This new polypeptide was namedsecretory DSS1 protein (sDSS1), the polypeptide sequences are expressedas follows:

DSS1 (Homo sapiens): (see SEQ ID NO: 4)MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNWDD DNVEDDFSNQLRAELEKHGYKMETSs-DSS1 (Homo sapiens): (see SEQ ID NO: 1)MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNWDDDNVEDDFSNQLRATVLLMILVCETPYGCYVLHQKGRMCSAFLCC

According to the screening of the sequenced primate genome and otheranimal pattern genomes in NCBI database, only the genome of Anthropoideaanimals has similar long mRNA sequence and polypeptide sequence similarto human sDSS1 protein, as shown in Table 1. The polypeptide sequencealignment results show that the sDSS1 protein sequence is highlyconservative, the N-terminal 59 amino acid sequences are identical, theother C-terminal amino acid sequences have a little point mutation (FIG.1B).

TABLE 1 Primate Species Amino acid sequence Haplorrhini HomoMSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNWDDDNVE sapiensDDFSNQLRATVLLMILVCETPYGCYVLHQKGRMCSAFL CC (see SEQ ID NO: 1) PanMSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW troglodytesDDDNVEDDFSNQLRATVLLMILVCETPYGCYVLHQKGRMCSAFL CC (see SEQ ID NO: 5) PanMSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW paniscus)DDDNVEDDFSNQLRATVLLMILVCETPYGCYVLHQKGRMCSAFL CC (see SEQ ID NO: 6)Gorilla MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW gorilla)DDDNVEDDFSNQLRVTVLLMILVCETLYGCYVLHQKGRMCSAFL CC (see SEQ ID NO: 7) PongoMSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW abeliiDDDNVEDDFSNQLRATILLMILVCETPYGCYVLHQKGRMCSAFLC C (see SEQ ID NO: 8)Nomascus MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW leucogenysDDDNVEDDFSNQLRATVLLMVLVCETPYGCYVLHQKERMCSAFL CC (see SEQ ID NO: 9)Rhinopithecus MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW roxellanaDDDNVEDDFSNQLRATVLLMIKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO: 10)Macaca MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW mulattaDDDNVEDDFSNQLRATVLLMIKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO: 11)Papio MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW anubisDDDNVEDDFSNQLRATVLLMIKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO: 12)Angola MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW colobusDDDNVEDDFSNQLRATVLLMKKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO: 13)Sooty MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW mangabeyDDDNVEDDFSNQLRATVLLMIKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO: 14)Mandrillus MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW leucophaeusDDDNVEDDFSNQLRATVLLMIKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO: 15)Macaca MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW nemestrinaDDDNVEDDFSNQLRATVLLMIKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO: 16)

The sDSS1 protein amino acid sequence was analyzed by using two kinds ofsecretory protein analysis and prediction software, which are Wolf PSORTand SecretomeP 2.0. The prediction results show that the sDSS1 proteinis located outside the cells, similar to multiple identified secretoryproteins, it is estimated as a secretory protein (Table 2). According tothe analysis result of Wolf PSORT software, the signal peptide cleavagesite of sDSS1 protein is located between amino acids positions 58-59.

TABLE 2 SecretomeP 2.0 WoLF PS ORT Predicted (Recommended threshold for(Numbers of similar protein Species Name secreted protein: 0.6) secretedproteins) location Homo sapiens 0.85 28 Extracellular Pan troglodytes0.85 28 Extracellular Pan paniscus 0.85 28 Extracellular Nomascusleucogenys 0.85 27 Extracellular Gorilla gorilla 0.752 23 ExtracellularPongo abelii 0.86 27 Extracellular Rhinopithecus roxellana 0.836 29Extracellular Macaca mulatta 0.836 29 Extracellular Angola colobus 0.82328 Extracellular sooty mangabey 0.836 29 Extracellular M. leucophaeus0.836 29 Extracellular Macaca nemestrina 0.836 29 Extracellular Papioanubis 0.836 29 Extracellular

According to the bioinformatic analysis results, the complete sequenceor C-terminal 31 amino acid sequences (31 amino acid sequences afteramino acid position 58) of the protein are connected to greenfluorescent protein (GFP) and expressed in 293T cells (sDSS1-GFP,sDSS1-c-GFP). The results show that the solution had green fluorescence,the background emitted light, and the fluorescence in the cells was dim.There was no fluorescence in the control group (GFP) solution, thebackground was very dark, the fluorescence in the cells was clear andbright (FIG. 2A). The cell culture medium was tested by point membraneimmunoblotting, the GFP signal was detected in the cell culture media ofsDSS1-GFP and sDSS1-c-GFP groups, but the signal was not detected in thecontrol group (GFP) (FIG. 2B). To sum up these results, the sDSS1protein is a sort of secretory protein, it can be synthesized in thecells and secreted out of the cells, the C-terminal 31 amino acidsequences of sDSS1 protein perform the function of signal peptide.

Example 2, sDSS1 Protein is a Naturally-Occurring Protein

1. Human serum and CSF sample treatment. Fresh human whole blood wascollected, kept still at room temperature for 10-20 minutes, 3500 gcentrifuged for 30 minutes, the supernatant was human serum. The serumwas mixed with 100 mM mercaptoethanol uniformly and treated by boilingwater bath for 10 minutes, 12000 g high speed centrifuged for 10 minutesafter cooling, the supernatant and ⅕ of 5× loading buffer solution byvolume were mixed. The fresh CSF was obtained from hospital and placedin ice box for transportation, treated on the day. The fresh CSF wasmixed with 5× loading buffer directly and made into samples directly forloading.

2. Western blotting, 15 μL prepared loading sample was put in theloading well, the protein was separated with 4-12% prefabricated gel(Life technology C#NP0321BOX) and moved to PVDF membrane. The membranewas subjected to primary antibody (Rabbit-anti-sDSS1) (antigen sequence:C-terminal 31 amino acid sequences of sDSS1 protein) incubation, PBSTsolution cleaning three times; and secondary antibody (Goat-anti-rabbitHRP antibody) incubation. It was cleaned with PBST three times,developed by luminescent liquid and the bands were displayed by X-rayfilm.

3. Cell culture, the human glioma cells (U87-MG cells) were bought fromATCC, the cells were cultured in complete cell culture medium containing90% basal medium DMEM and 10% FBS, cultured in cell incubator(temperature 37° C., humidity 95%, CO2 concentration 5%), subculturedonce every two days.

4. PCR experiment, the U87-MG cells were collected and lysed rapidly,the total RNA was extracted from cell lysis solution by using a totalRNA extraction kit (QIAGEN,51304), the RNA sample was treated with 1U/μL DNase I at room temperature for 15 minutes to remove residualgenome DNA. The obtained RNA sample was all converted and synthesizedinto cDNA by using a cDNA synthesis kit (TransGen Biotech, AT301) andused as template sample for subsequent PCR experiment. 20 μL reactionsystem was used in the PCR reaction, including 100 μL, PCR premixedreagent (PCRTaq Mixture) (Omega bio-tek, TQ2200), 0.54, cDNA template(3.5 μg/mL), 0.54, primer, 94, ultrapure water, mixed uniformly beforePCR reaction. DSS1 cDNA primers: forward primer: GCAGACAGTCGAGATGTCAGAG,reverse primer: TTCTTCTGGATGCTATGAAGTCTCC; sDSS1 cDNA primers: forwardprimer: GCAGACAGTCGAGATGTCAGAG, reverse primer: TGATGATCTGTTAACAGCAGAGG.PCR reaction procedure: 94° C. 10 minutes, cyclic reaction 40 times:including 94° C. 10 s, 62° C. 20 s, 72° C. 20 s, 72° C. 10 minutes afterthe circulation is finished, stored at 4° C. and the DNA content in PCRproduct was tested by 3% sepharose [0.05% SYBR Green Stain (ThermoFisher, 4472903)] electrophoresis.

Result Analysis:

The signal of sDSS1 protein can be detected in CSF or serum by using thespecific antibody of sDSS1. The serum samples derived from differentindividuals manifest different signal modes, the sDSS1 signal in theserum of the individual after exercise was apparently higher than thatof the individual in resting state (FIG. 3A). The mRNA signal of sDSS1gene can be detected in U87-MG cells, the gene sequencing result of PCRamplified product was identical to the sequence of database (FIG. 3B).These results show that the sDSS1 protein is a sort of protein, existingin CSF and serum.

Example 3, Small-Amount Preparation of sDSS1 Protein Experimental Method

1. SDSS1 protein preparation: the nucleotide segment of total genesynthesis coded human sDSS1 protein (see SEQ ID NO: 17) was insertedinto the back of Hisx6-V5 tag in pET151D. The plasmid was transferred tothe expression strain BL21 (DE3). The Escherichia coli was fused withexpression Hisx6-V5-sDSS1 protein, the Ni-NTA gel column was used forpreliminary purification, and then the SDS-PAGE was used for gelpurification. The cut strip containing His-V5-sDSS1 protein was put inthe bag filter with transfer buffer. The protein was removed from thegel under the drive of electric field and collected in the bag filter.The protein was concentrated to about 500 dialyzed in PBS solution at 4°C. four times, 200 ml each time.

2. SDS polyacrylamide gel electrophoresis, the purified sDSS1 protein orbacterial lysis solution protein was mixed with 5× loading buffersolution, treated by boiling water bath for 10 minutes, 12000 g highspeed centrifuged for 10 minutes, the supernatant was extracted foranalysis. The protein was separated by 4-12% prefabricated gel, the gelwas stained for 1 hour using Coomassie brilliant blue staining solution,and decolored by destainer at room temperature over night. When thedecolorization was completed, the bands on the gel were observed andphotographed.

Result Analysis:

The positive cloned Escherichia coli strain was selected, the culturewas expanded, the bacterial cells were stimulated by IPTG to expressobjective protein at the beginning of logarithmic phase of bacterialgrowth. The bacteria were lysed, the objective protein expression levelwas tested. The result shows after the IPTG stimulation, the sDSS1protein expression level of bacterial cells was upgraded significantly.The protein bands were obvious in the gel image (FIG. 4A). After thebacterial lysis solution was preliminarily purified by Ni-NTA gelcolumn, the objective protein in concentrate was concentrated greatly(channel b), the impure protein content decreased, very pure sDSS1protein could be obtained by further purification (channel a)(FIG. 4B),applicable to subsequent bioexperiment. The purified sDSS1 protein wasquantified by BCA protein, the final concentration was 0.72 mg/ml,stored at 4° C. for future use.

Example 4, sDSS1 Protein Reacts with Oxidized Protein and ShieldsCytotoxicity of Oxidized Protein Experimental Method

1. Reaction between oxidized serum and sDSS1 protein, 3500 g of freshblood was centrifuged for 30 minutes, the upper serum was extracted forsubsequent experiment. The 10 μL sDSS1 protein solution (0.72 mg/mL) wasmixed with 10, 20, 50 and 1004, oxidized serums respectively, the massratios of sDSS1 to serum protein were about 1:100, 1:200, 1:500 and1:1000, mixed with 20 μM Fenton reagent (FeSO₄ and H₂O₂ were mixed asper mass ratio of 1:1), incubated in a dark place at 4° C. over night.On the next day, the reacting His-V5-sDSS1 was separated by using 10 μLNi-NTA beads. The reactant liquor was mixed with the beads at 4° C. for2 hours, the magnetic separation device adsorbed the beads on the tubewall, the liquid was removed, 1 ml PBST was applied, the tube wasremoved from the magnetic separation device, after repeated oscillationcleaning, the magnetic separation device adsorbed the beads, the PBSTwas sucked away, and the above steps were repeated four times. Finally,the protein was eluted with 504, TBS containing 50 mM EDTA, the eluentwas mixed with isometric 2×SDS solution, treated at 100° C. for 10minutes, 12000 g centrifuged for 10 minutes, the supernatant wasextracted for test. The supernatant was mixed with 5× loading buffersolution, heated at 100° C. for 10 minutes, the prepared sample was usedfor western blotting.

2. Preparation of oxidized FBS and AOPP, 10 mL FBS was mixed with 10 mMNaC10 and treated for 1 hour, the oxidized serum was dialyzedcontinuously in PBS solution using 3000 Da bag filter for 24 hours, thesolution was changed at intervals of 8 hours during dialysis, thetreated serum solution was mixed with 1 mM vitamin C (Vc) to remove theparticipant oxidizer completely. The protein concentration was tested byBCA protein quantification. The content of oxidized protein was testedby using two methods, the dityrosine value in the oxidized serummeasured by chloramine-T was 75.31 μM/mg protein (untreated serum was15.05 μmol/mg protein), the carbonyl content detected bydinitrophenylhydrazine was 16.33 nmol/mg protein (untreated serum was13.68 nmol/mg protein).

The 10 mg serum albumin was treated with 160 mM NaC10 for 1 hour, theoxidized protein was dialyzed continuously in PBS solution using 3000 Dabag filter for 24 hours, the solution was changed at intervals of 8hours during dialysis. The protein concentration of the treated AOPP wasdetermined by BCA protein quantification. The dityrosine value of AOPPsample measured by chloramine-T was 54.21 μmol/mg protein (untreated BSAwas 14.55 μmol/mg protein), the carbonyl content measured bydinitrophenylhydrazine was 1042.57 nmol/mg protein (untreated BSA was10.26 nmol/mg protein).

3. Reaction between AOPP and sDSS1 protein, the 1504, reaction systemwas mixed with 30 μg AOPP protein (200 μg/mL), and mixed with 15 μg (100μg/mL), 30 μg (200 μg/mL) and 60 μg (400 μg/mL) sDSS1 proteinrespectively, the excess volume was supplemented by aseptic PBSsolution. The solution was stirred uniformly and reacted at 4° C. overnight. The sample after reaction was mixed with 5× loading buffersolution, heated at 100° C. for 10 minutes, the treated sample wasseparated by SDS-PAGE and the bands were displayed by Coomassiebrilliant blue staining.

4. Western blotting, the protein mixture after reaction was mixed with5× loading buffer, treated by boiling water bath for 10 minutes forwestern blotting analysis. The specific method was the same as describedabove. The antibody was V5-HRP antibody (1:5000 diluted).

5. Cell line culture, the human neuroblastoma cells (SH-SYSY) were grownin the basal medium DMEM with 10% FBS; the cells were subcultured onceevery two days.

6. Cell viability test, in order to test the effect of sDSS1 protein onthe cytotoxicity of oxidized serum, the SH-SYSY cells were inoculated toa 96-well plate as per 10⁴ cells per well, 2000, complete medium. 12hours later, the complete medium was changed to DMEM without serumcontaining 0.5% BSA 2004, per well. After 24 hours of treatment, theDMEM solution was changed to 10% oxidized serum and 10% oxidized serumcontaining 20 μg/mL sDSS1 protein as culture medium, 2004, per well.After 48 hours of treatment, the old culture medium was removed from the96-well plate, 1004, diluted CCK-8 working fluid (1:20 diluted)(DOJINDO, CK04) was put in each well to test the changes in cellviability. The group with BSA at isoconcentration was the control group.

In order to test the protective effect of sDSS1 protein on thecytotoxicity induced by AOPP, the SH-SYSY cells were inoculated to the96-well plate as per 2×10⁴ cells per well, after 12 hours of adhesion,the culture medium was changed to culture medium without serumcontaining 0.5% BSA. After 24 hours of treatment, it was changed toculture medium without serum containing 100 μg/mL AOPP protein, and thetreatment group was provided with 100 μg/mL sDSS1 protein, 2004, perwell. After 48 hours of treatment of 96-well plate, the changes in cellviability were tested by using CCK-8 kit.

Result Analysis:

The serum contained a lot of proteins, mainly being serum albumin. Underthe effect of Fenton reagent, the proteins in serum were oxidized, theoxidation products reacted with sDSS1 to foam complexes. In controlgroup, the sDSS1 protein monomer had no obvious protein aggregation. Inthe experimental group, the co-incubation with serum led to theformation of lots of high molecular weight protein complexes, thesecomplexes cannot be separated by SDS-PAGE (FIG. 5A). The result showsthat the sDSS1 protein can combine with the oxidized protein in serum.In the cytotoxicity experiment, compared with control serum, theaddition of 10% oxidized protein could depress cell proliferation andcell viability obviously, and 20 μg/mL sDSS1 protein in the culturemedium could retrieve the cytotoxicity of oxidized protein (FIG. 5B).

The sDSS1 was mixed with AOPP, the sDSS1 and AOPP were combined to formcomplexes, these complexes cannot be separated by SDS-PAGE. The numberof complexes increased apparently with the sDSS1 protein concentrationin the reaction system (FIG. 5C). In the cell experiment, the AOPP hadsignificant cytotoxicity for cells, reducing the cell viability, and thesDSS1 at isoconcentration could shield the cytotoxicity of AOPPcompletely (FIG. 5D). To sum up the results, the sDSS1 can protect thecells from the cytotoxicity of oxidized serum or AOPP.

In addition, to sum up the reaction between sDSS1 protein and oxidizedprotein, two proteins can combine with the oxidized protein tightly,this binding force can resist high concentration of SDS, which seems tobe covalent interaction. The difference is that the combination processof DSS1 and oxidized protein needs the assistance of an ATP enzyme[Zhang et al, 2014]. Our evidence shows that the tight coupling of sDSS1and oxidized protein is free of ATP, the ATP enzyme is not required.According to the amino acid sequences of DSS1 and sDSS1, the sequencesof amino acid positions 1 to 58 of the two proteins are identical, andthe sequence of amino acid positions 59 to 70 of DSS1 are completelydifferent from the sequence of amino acid positions 59 to 89 of sDSS1.The tight coupling of DSS1 and sDSS1 with oxidized protein is supposedto be derived from the shared amino acid sequences, i.e. the sequence ofthe first 58 amino acids. The difference in characteristic between sDSS1and DSS1, which is the characteristic that the tight coupling withoxidized protein is free of ATP enzyme mediation, should be derived fromthe unique amino acid sequence of sDSS1, i.e. C-terminal amino acidsequence of positions 59 to 89. Altogether, the tight coupling withoxidized protein without ATP enzyme mediation of sDSS1 is derived fromthe organic combination of the sequences of the first 58 amino acids andthe last 31 amino acids.

Example 5, sDSS1 Protein Reduces the Formation of Aβ Oligomer andReduces the Cytotoxicity of Aβ Oligomer Experimental Method

1. Cell line culture, the human neuroblastoma cells (SH-SY5Y) were grownin the basal medium DMEM with 10% FBS; the cells were subcultured onceevery two days.

2. Neural stem cell culture, the neural stem cells (NSCs) were from P2mouse brain tissue, the NSCs of primary suspension culture were used fortoxicity test after two subcultures, the NSCs were cultured in the stemcell culture medium, including 88% DMEM/F12 basal medium (Gibco,C#12500-062), 10% Proliferation supplementary additive (Stem celltechnology, C#05701), 2% BSA (Sigma, C#V900933), 10 ng/mL Heparin(Sigma, C#H3149), 10 ng/mL bFGF (Roche, C#11104616001), 20 ng/mL EGF (BDBioscience, C#354010).

3. Reaction between Aβ and sDSS1 proteins, the Aβ protein (Human, 1-42)freeze-dried powder was supplied from Suzhou Qiangyao Biotechnology Co.,Ltd. 2 mg Aβ freeze-dried powder was dissolved by 20 μL DMSO, dilutedwith PBS to 2 mg/mL, stored at −20° C. The reaction system was providedwith 300 μL PBS solution the 10 μg Aβ and sDSS1 proteins were mixed asper molar mass ratios 1:1, 1:5 and 1:10, and then incubated at 4° C.over night. The incubated reactant was mixed with 5× loading buffersolution, treated at 100° C. for 10 minutes for western blottinganalysis.

4. Aβ protein pretreatment, the Aβ stock solution was diluted with basalmedium (pH7.2) to 1000 μg/mL, the Aβ diluent was incubated at 4° C. for24 hours to form oligomer for cell experiment. The Aβ concentration insubsequent experiment was always labeled according to the proteinconcentration before incubation.

5. Western blotting, the treated reactant was separated by SDS-PAGE forwestern blotting analysis, the specific method was the same as describedabove. The antibodies used were V5-HRP antibody (1:5000 diluted), Aβantibody (Cell signal technology, 9888), secondary antibody(Goat-anti-rabbit HRP antibody).

6. Cell viability test, the SH-SY5Y cells were inoculated to the 96-wellplate as per 2×10⁴ cells per well, after 12 hours of adhesion, the oldculture medium was changed to culture medium without serum containing0.5% BSA, after 24 hours of treatment, the old culture medium waschanged to DMEM solution containing Aβ or Aβ and sDSS1 proteins. After48 hours of treatment of cells, the cell viability level was tested byCCK-8 kit.

7. Cell apoptosis test, the cell apoptosis test kit was bought fromDOJINDO chemical technology (Shanghai) corp. (AD10). The SH-SY5Y cellswere inoculated to the 6-well plate as per 3×10⁵ cells per well, after12 hours of adhesion, the old culture medium was changed to DMEMsolution without serum containing 0.5% BSA. After 24 hours of treatment,it was changed to solution containing Aβ or Aβ and sDSS1 proteins. After48 hours of treatment, the cell apoptosis level was tested by apoptosiskit. All of the solution and cells were collected, the supernatant wasremoved by centrifugation. The cells were resuspended in the 4004,staining buffer solution provided by the apoptosis kit, 185 μL cellsuspension was extracted for subsequent test. The cell suspension wasmixed with 54, Annexin V staining solution uniformly, the cells wereincubated at 37° C. for 10 minutes. It was mixed with 10 μL PI stainingsolution uniformly, the cell apoptosis level was tested by flowcytometer.

The NSCs were firstly adhered and cultured in the 6-well plate, theplate was treated with 0.025% Laminin for at least two hours and cleanedwith aseptic PBS 6 times for future use. The NSCs were made intounicells and inoculated to 6-well plate as per 3×10⁵ per well, the cellswere adhered for 24 h for subsequent experiment.

Result Analysis

The Aβ protein had obvious aggregation after incubation, there wereprotein aggregates of different sizes formed within 10-20 KD. Accordingto previous reports, these Aβ oligomers were the main source of the Aβinduced cytotoxicity. After co-incubation of sDSS1 protein and Aβ, thesDSS1 protein and Aβ protein aggregated to form high molecular weightcomplex (molecular weight higher than 20 KD), the oligomers formedwithin 10-20 KD were reduced obviously (FIG. 6A). As the sDSS1 proteinconcentration increased, the formation of Aβ oligomer was depressedapparently. According to the sDSS1 protein signal detection, the complexwas formed by the reaction between sDSS1 protein and Aβ (FIG. 6B), andit could not be separated by SDS-PAGE.

The shielding effect of sDSS1 protein on the Aβ induced cytotoxicity wastested. In the cell viability test, the cell viability declinedsignificantly after the pretreated Aβ oligomer was applied. When theculture medium was mixed with sDSS1 protein, the SH-SY5Y cell viabilitywas recovered significantly and the cell viability was higher thancontrol group (FIG. 6C). In the cell apoptosis test, the addition of Aβoligomer to the culture medium induced the apoptosis of SH-SY5Y cells orNSCs. The addition of sDSS1 protein to the culture medium can reduce theearly apoptosis and late apoptosis levels of cells significantly (FIG.6D, FIG. 6E). According to the results, the sDSS1 protein can combinewith Aβ protein to reduce the Aβ oligomer formation, so as to mitigatethe cytotoxicity induced by Aβ protein.

Example 6 sDSS1 Protein Reduces Cytotoxicity of Amylin OligomerExperimental Method

1. Cell line culture, the human neuroblastoma cells (SH-SY5Y) were grownin the basal medium DMEM with 10% FBS; the cells were subcultured onceevery two days.

2. Amylin protein pretreatment, the amylin protein (Human) freeze-driedpowder was supplied from Suzhou Qiangyao Biotechnology Corp. The 2 mgamylin freeze-dried powder was dissolved to 2 mg/mL in 10 mM sodiumacetate solution (pH5.5), stored at −20° C. The amylin stock solutionwas diluted to 1 mg/mL with basal medium (pH7.2). The amylin diluent wasincubated at 4° C. for 48 hours to form oligomer for cell experiment.The amylin concentration in subsequent experiment was always labeledaccording to the protein concentration before incubation.

3. Cell viability test, the SH-SY5Y cells were inoculated to 96-wellplate as per 2×10⁴ cells per well, after 12 hours of adhesion, the oldculture medium was changed to culture medium without serum containing0.5% BSA. After 24 hours of treatment, the old culture medium wasremoved and the DMEM solution containing amylin or amylin and sDSS1proteins was applied. After 48 hours of treatment of cells, the cellviability level was tested by CCK-8 kit.

Result Analysis

The addition of 10 μM inbubated amylin protein to the cell culturemedium can induce significant cytotoxicity, and the addition of sDSS1protein can shield the cytotoxicity induced by amylin oligomer, the cellviability was even higher than control group (FIG. 7), meaning the sDSS1protein can shield the cytotoxicity of amylin protein effectively.

Example 7 sDSS1 Protein Reduces Cytotoxicity of Glycosylated ProteinExperimental Method

1. Cell line culture, the human neuroblastoma cells (SH-SY5Y) were grownin the basal medium DMEM with 10% FBS; the cells were subcultured onceevery two days.

2. Glycosylated protein preparation, 10 mg/mL serum albumin and 2.5Mribose were mixed and incubated at 37° C. for 7 days, and then dialyzedin PBS using 3000 Da bag filter for 24 hours, the solution was changedat intervals of 8 hours. The completed glycosylated protein wasquantified by BCA, the sample was stored at −80° C. for future use.

3. Cell viability test, the SH-SY5Y cells were inoculated to 96-wellplate as per 2×10⁴ cells per well, after 12 hours of adhesion, the oldculture medium was changed to culture medium without serum containing0.5% BSA. After 24 hours of treatment, the old culture medium wasremoved and the DMEM solution containing glycosylated protein orglycosylated protein and sDSS1 proteins at different concentrations wasapplied, the group with BSA at isoconcentration was used for control.After 48 hours of treatment of cells, the cell viability level wastested by CCK-8 kit.

Result Analysis

The addition of 400 μg/mL glycosylated protein to the cell culturemedium can induce significant cytotoxicity, and the addition of sDSS1protein can reduce the cytotoxicity induced by glycosylated protein. Asthe sDSS1 protein concentration increased, the cell viability was evenhigher than control group (FIG. 8), meaning the sDSS1 protein can shieldthe cytotoxicity of glycosylated protein effectively.

Example 8, sDSS1 Protein Prolongs Postoperative Survival Time ofSenescence-Accelerated Mouse SAMP Experimental Method

1. Animal feeding, the senescence-accelerated SAMP8 mice (5 months old,male) were bought from Beijing Vital River Laboratory Animal TechnologyCo., Ltd., the animals were fed at the clean laboratory animal breedingcenter of Southern Model Organism Center. The animals were provided withsufficient aseptic water and standard mouse breeding feed, 12 h/12 hdark-and-bright alternate illumination, the bedding and cage werechanged monthly, the animal survival was observed daily.

2. Adenovirus synthesis, the nucleic acid sequence (see SEQ ID NO: 17)of adenovirus expressing sDSS1 protein was provided by us (inventors),the adenovirus construction and synthesis were completed by Cyagen(Guangzhou) Biotechnology Co., Ltd. The adenovirus promoter andtranscription region sequence composition: pAV[Exp]-UBC>EGFP:T2A:ORF_363bp, including ubiquitin protein promoter sequence. According todetermination, the virus titer was larger than 10¹⁰ PFU/mL. According tothe validation by infecting U87-MG cells and mouse neuroblastoma cells(N2a), the adenovirus can infect cells and express sDSS1 proteinefficiently. The adenovirus expressing GFP protein (pAV[Exp]-UBC>EGFP)was used as control.

3. Stereotactic injection, the senescence-accelerated SAMP8 mouse (6months old, male) was anaesthetized by intraperitoneal injection with20% urethane (dissolved in normal saline, impurities and bacteriaremoved by 0.22 μm filter) as per 800 mg/kg body weight. When the mousewas anaesthetized, it was fixed to the mouse stereotaxic apparatus(Stoelting, 51500), the cranial bone was kept horizontal. The head skinwas incised to expose the cranial bone, the bregma was taken as thestarting coordinate to locate the lateral ventricle region (0.58 mm,1.25 mm, 1.75 mm). The marking point was perforated by dental drill. 24,of virus liquid was sucked by the microinjector (Hamilton 600-2.5 μL,syringe needle diameter 0.2 mm), the lateral ventricle region wasrelocated according to the same coordinate values. The needle wasinserted into the lateral ventricle region quickly according to thespecified coordinate values, the virus liquid was injected slowly at 200nL/min on average, for a total amount of 1000 nL. After the injectionwas done, each time the needle was lifted for 0.25 mm, it was waited for3 minutes until the needle was drawn out of the brain tissue completely.The lateral ventricle region on the opposite side was relocated, theneedle insertion, injection and needle lifting were completed, 1 μLvirus liquid was injected. For the mouse after injection, the cranialbone and peripheral tissues were wiped with 100 U/mLampicillin/streptomycin, and the skin was sutured. The abdominal cavityof the mouse was injected with 150 μL antibiotics, and the mouse was putin the cage with abdomen up until the mouse was awake.

Result Analysis

The schematic diagram indicates the basic operation of injecting virusinto the mouse's lateral ventricle and the adenovirus injection site(FIG. 9A). Wherein there were two batches of mice injected withadenovirus expressing sDSS1, 10 mice in total; there were two batches ofmice injected with adenovirus expressing GFP, 14 mice in total. Within 1month after the injection of adenovirus, three mice of the control groupdied, and one mouse of the experimental group died. Within 8 months, themice of the control group died successively, whereas only one mouse ofthe experimental group died (FIG. 9B). To sum up the results, thepostoperative survival time of the SAMP8 mice injected with adenovirusexpressing sDSS1 protein was significantly longer than that of the miceonly injected with control virus. The significant difference wasanalyzed by ANOVA (P-value<0.01).

The above detailed description only specifies the feasible embodimentsof the present application and is not intended to limit the scope ofprotection of the present application. Any equivalent embodiments oralterations not deviating from the gist of the present application shallbe covered in the scope of protection of the present application.

1-18. (canceled)
 19. A composition comprising: (i) a polypeptide havingan amino acid sequence at least 80% identical to any one of SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8,SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO.13, SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16; or (ii) a nucleicacid encoding said polypeptide of (i), which composition has beenformulated for administration to a subject.
 20. The composition of claim19, wherein the polypeptide comprises an amino acid sequence having atleast 80% sequence identity to SEQ ID NO:
 1. 21. The composition ofclaim 19, wherein the polypeptide comprises the amino acid sequence ofSEQ ID NO:
 1. 22. The composition of claim 19, wherein the nucleic acidcomprises a polynucleotide sequence at least 80% identical to SEQ ID NO:17.
 23. The composition of claim 19, wherein the polypeptide comprises afusion protein.
 24. The composition of claim 23, wherein the fusionprotein comprises the polypeptide fused to itself or a fragment thereof.25. The composition of claim 23, wherein the fusion protein comprisesthe polypeptide fused to a carrier protein.
 26. The composition of claim23, wherein the fusion protein comprises the polypeptide fused to anantibody.
 27. The composition of claim 19, wherein the polypeptide iscomplexed with a pharmaceutically acceptable carrier.
 28. Thecomposition of claim 27, wherein the pharmaceutically acceptable carriercomprises one or more of microspheres, vesicles, liposomes,microemulsions, nanoparticles, magnetic particles, or gels.
 29. Thecomposition of claim 19, wherein the polypeptide further comprises apharmaceutically acceptable excipient.
 30. The composition of claim 19,wherein the polypeptide is complexed with a pathogenic polypeptideselected from the group consisting of advanced oxidation proteinproducts (AOPP), amyloid-β, amylin, and glycosylated protein.
 31. Thecomposition of claim 30, wherein the pathogenic polypeptide is advancedoxidation protein products (AOPP).
 32. The composition of claim 30,wherein the pathogenic polypeptide is amyloid-β.
 33. The composition ofclaim 30, wherein the pathogenic polypeptide is amylin.
 34. Thecomposition of claim 30, wherein the pathogenic polypeptide isglycosylated protein.
 35. The composition of claim 19, wherein thepolypeptide is complexed with a pathogenic polypeptide, wherein thepathogenic polypeptide is glycated protein.
 36. A method for treating orpreventing dementia in a subject, the method comprising administering acomposition of claim 19 in an effective amount to the subject in needthereof.
 37. The method of claim 36, wherein the dementia is Alzheimer'sDisease (AD).
 38. A method for treating or preventing type II diabetesin a subject, the method comprising administering a composition of claim19 in an effective amount to the subject in need thereof.